Human Plasma Fibrinogen Adsorption on Ultraflat Titanium Oxide Surfaces Studied with Atomic Force Microscopy

Abstract

Conformational changes of proteins adsorbing on biomaterial surfaces affect biocompatibility. Titanium is among the most successful biomaterials; however, the mechanisms leading to its biocompatibility are not yet understood. The primary objective of this study was to investigate the conformation of human plasma fibrinogen (HPF) adsorbed on titanium oxide surfaces. A method for preparing ultraflat titanium substrates was developed. This allowed high-resolution investigation of both the titanium oxide surfaces and the adsorbed state of HPF. The titanium oxide surfaces were first imaged with an atomic force microscope in air and subsequently incubated in a solution of HPF in phosphate buffer and imaged in fluid with tapping mode AFM. The titanium oxide surfaces exhibited a root-mean-squared (RMS) roughness of (0.29 ± 0.03) nm over (1.00 × 1.00) μm2 areas. Different degrees of molecular order were found on the titanium oxide surface. In crystalline surface areas square lattices with parameters a 0 = b 0 ≈ 0.5 nm were resolved, which is consistent with the (001) planes of the titanium dioxide TiO2 rutile. Repetitive scanning of TiO2 surface areas resulted in the abrasion of the outermost TiO2 layers by the AFM tip to a depth varying between 0.3 and 1.0 nm. When imaging adsorbed HPF, individual molecules and aggregates, often joined through their D domains, were resolved, and the typical HPF multiglobular structure was observed. The mean length and height of single molecules were (46 ± 3) nm and (1.4 ± 0.2) nm, respectively. Additional features (chain segments) adjacent to the D and E domains were resolved and attributed to the α chains and their C-termini. The combination of AFM and the ultraflat titanium preparation method has proven successful for the high-resolution study of both the TiO2 surface and the adsorbed HPF.

Protein Adsorption on Nano-scaled, Rippled TiO2 and Si Surfaces

“…A general understanding of the mechanisms involved in protein adsorption can be gained by investigating human plasma fibrinogen (HPF) on surfaces, because of its importance for the blood coagulation process and the fact that its structure is well known [1,7,8]. Most notably, HPF has an amphiphilic character that causes its hydrophobic and hydrophilic parts to be attracted to hydrophobic and hydrophilic surfaces, respectively, assuming different conformations.…”

“…Surface-bonded HPF has a length of 46-49 nm and a height about 0.3-2.5 nm depending on the protein conformation on different substrates and under different environmental conditions [7,[10][11][12][13]. Former studies mostly investigated the adsorption of HPF and a variety of other proteins [14,15] on flat substrates such as commercially pure Ti with a natural TiO 2 layer on top [13], TiO 2 [7], graphite [10,11,16], mica [10,12,13], ultra-high molecular weight polyethylene [17], Si [18] or SiO 2 [19]. In most cases, these investigations concentrated on the adsorption of single proteins in air and/or under aqueous conditions.…”

Formation and cross-linking of fibrinogen layers monitored with in situ spectroscopic ellipsometry

“…During the last decades numerous investigations have been performed dealing with adsorption from single protein solutions as well as binary and ternary protein solutions where both steady-state and/or dynamics of adsorption have been monitored by a variety of techniques, e.g. 125 I radiolabeling [1] , circular dicroism [2], ellipsometry [3], reflectometry [4], AFM [5] and intrinsic fluorescence measurements. Most of the work has been focused on presentation of thickness and/or surface mass density of protein films in steady-state.…”

Cell-Protein-Material Interaction in Tissue Engineering

“…a technique able to provide direct observation of protein conformation on different substrates has been extensively used in recent years, mainly on model surfaces, such as silica, mica, titanium graphite, and self-assembled monolayers (SAMs), flat enough so that the height magnitude is able to reveal the trinodular structure of single-adsorbed FG molecules (Toscano & Santore, 2006;Marchin & Berrie, 2003;Agnihotri & Siedlecki, 2004;Cacciafesta et al, 2000;Tunc et al, 2005;Gettens et al, 2005;Gettens & Gilbert, 2007;Ta & McDermott, 2000;Ishizaki et al, 2007;Ortega-Vinuesa et al, 1998;Mitsakakis et al, 2007;Sit & Marchant, 1999). The effect of surface wettability, as one of the most important parameters that affects the biological response to a material, on FG adsorption has lead to different, nonconsistent conclusions.…”